The role of exocyst members sec6 and sec8 in the anterograde trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells
In epithelia, ion channels play an essential role in maintaining the required electrochemical gradients for the tissue to function. This function can translate to electrolyte and fluid homeostasis which in turn, relies on ion channels being targeted to respective apical or basolateral membranes (BLM). KCa3.1, the intermediate conductance, calcium activated potassium channel, is one such channel which is targeted to the BLM where it facilitates absorption and secretion. Over the last two decades, an increasing body of research has suggested that altered BLM populations of KCa3.1 may result in clinical manifestation of diseases. Other proteins targeted to the BLM rely on the exocyst complex to facilitate arrival at the BLM, and two exocyst proteins (sec6 and sec8) are key to traffic cargo specifically to the BLM. Therefore, this study aimed to investigate if sec6 and sec8 are required for trafficking of KCa3.1 to the BLM, and if the respective sec proteins directly interact with KCa3.1. To investigate, a Fischer rat thyroid cell line that stably expresses KCa3.1 was used as an epithelial model. The cells underwent reverse transfection with short interfering RNA (siRNA) against sec6 and sec8. Ussing chamber experiments were conducted with KCa3.1 specific agonist 1-EBIO and antagonist clotrimazole (CLT) to measure KCa3.1 specific Potassium (K+) current as a functional measure of KCa3.1. Immunoblotting was used to measure the BLM population of KCa3.1 and lastly Co-immunoprecipitation (Co-IP) was performed to test if sec proteins interact with KCa3.1. Immunoblot experiments revealed there were significant decreases of KCa3.1 expression at the BLM by 54±1 % (p <0.001; n=3) when sec6 was knocked down and by 76±11 % (p<0.01, n=3) when sec8 was knocked down. Ussing chamber showed a 97±0.1 % (P<0.001, n=5) reduced 1-EBIO-elicited K+ current as well as an 88±12 % (P<0.01, n=5) reduced CLT-inhibited K+ current when sec6 was knocked down. Similarly, knock down of sec8 resulted in a 91±10 % (P<0.001, n=5) reduced 1-EBIO elicited K+ current as well as a 44±16 % (P<0.05, n=5) reduced CLT inhibited K+ current. Co-iP experiments only investigated any potential interaction between sec6 and KCa3.1 which was not seen (n=3). Based on the immunoblot, Ussing chamber data and Co-iP results, sec6 and sec8 are both important for anterograde trafficking of KCa3.1 to the BLM of a polarised epithelium, and that the potential interaction is likely to be happening between sec8 and KCa3.1. Collectively, these results advance our knowledge of anterograde KCa3.1 trafficking in polarised epithelial tissue, as well as the importance of exocyst members sec6 and sec8 in the process of anterograde trafficking of BLM destined cargo. Moreover, this may provide an avenue for novel drug targets for KCa3.1 associated pathologies, especially those associated with increased or decreased BLM populations of KCa3.1.
Advisor: McDonald, Fiona; Hamilton, Kirk
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: Physiology
Publisher: University of Otago
Keywords: Epithelia; Trafficking; KCa3.1; SK4; Gardos; IK1; KCNN4
Research Type: Thesis